Syddansk Universitet
Heat hardening as a function of developmental stage in larval and juvenile Bufo
americanus and Xenopus laevis
Sherman, Elizabeth; Levitis, Daniel
Published in:
Journal of Thermal Biology
Publication date:
2003
Citation for pulished version (APA):
Sherman, E., & Levitis, D. (2003). Heat hardening as a function of developmental stage in larval and juvenile
Bufo americanus and Xenopus laevis. Journal of Thermal Biology, 28(5), 373-380.
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ARTICLE IN PRESS
Journal of Thermal Biology 28 (2003) 373–380
Heat hardening as a function of developmental stage in larval
and juvenile Bufo americanus and Xenopus laevis
Elizabeth Sherman*, Daniel Levitis
Natural Sciences, Bennington College, Bennington, VT 05201, USA
Received 26 October 2002; accepted 10 January 2003
Abstract
1. We studied heat hardening in tadpoles and juveniles of the temperate Bufo americanus and the subtropical Xenopus
laevis by comparing the critical thermal maxima (CTMax) of heat-pretreated animals with the CTMax of control
(non-pretreated) animals of the same developmental stages.
2. While premetamorphic tadpoles (Gosner stages 26–43) of both species exhibited heat hardening, metamorphosing B.
americanus tadpoles (stages 44–45) were unable to heat harden and, in fact, exhibited a decrease in CTMax following
pretreatment while metamorphosing X. laevis tadpoles still exhibited heat hardening.
3. This difference may reflect the more stressful nature of the more extensive metamorphosis of Bufo compared to that
of Xenopus.
r 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Tadpoles; Heat hardening; CTMax; Anuran development; Bufo; Xenopus; Thermal tolerance
1. Introduction
Vertebrate ectotherms exhibit elevated levels of
thermal tolerance following a brief, sublethal thermal
stress. This phenomenon, heat hardening, is an acute
short-lived response that permits animals to cope with
transient fluctuations in environmental temperature and
appears to be adaptive. Among amphibians, heat
hardening is particularly significant given the fluctuations in daily temperature to which they may be exposed
(Spotila et al., 1989; Rome et al., 1992). The thermal
environment of anuran amphibians changes during their
lifetime as most begin life as aquatic organisms which
later metamorphose into terrestrial or semi-terrestrial
animals (Sherman, 1980; Ultsch et al., 1999). Moreover,
both the thermal tolerance of tadpoles (Cupp, 1980;
*Corresponding author. Tel.: +1-802-440-4466; fax: +1802-440-4461.
E-mail address: [email protected] (E. Sherman).
Sherman, 1980; Floyd, 1983) and their selected temperatures (Dupre and Petranka, 1985; Wollmuth and
Crawshaw, 1988) change during their ontogeny. Therefore, we predicted that the heat hardening capacity of
tadpoles might also change during their development.
In this study, we examined the heat hardening
capacity of tadpoles and juvenile toads of Bufo
americanus and Xenopus laevis. Heat hardening was
measured as an increase in the critical thermal maximum
(CTMax) of animals that had been pretreated with a
thermal stress compared to the CTMax of nonpretreated control animals.
The thermal physiology of anurans is related to their
geographic distribution (Ultsch et al., 1999) and therefore we might expect differences in heat hardening
between the temperate B. americanus and the subtropical X. laevis. Amphibians from temperate climates
typically experience more extreme temperature fluctuations than tropical and subtropical forms (Ultsch et al.,
1999). Moreover, Bufo tadpoles can be found in
0306-4565/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0306-4565(03)00014-7
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374
E. Sherman, D. Levitis / Journal of Thermal Biology 28 (2003) 373–380
temporary ponds, often exploiting high temperatures
which encourages metamorphosis (Sherman, 1980),
while Xenopus typically inhabits permanent ponds and
has a more protracted metamorphosis (Wassersug and
Hoff, 1982).
Wassersug (1996) has argued that pipids, such as
X. laevis, which remain aquatic following transformation exhibit a less abrupt and extensive metamorphosis
than bufonids which transform rapidly into terrestrial
toadlets. A number of studies have reported a decrease
in thermal tolerance of anuran larvae undergoing
metamorphosis (Cupp, 1980; Sherman, 1980; Floyd,
1983). Sherman (1980) has suggested that such a
decrease is due to the stressful nature of metamorphosis
which requires extensive differentiation. If Bufo has a
more extensive, abrupt, and therefore stressful metamorphosis than Xenopus, perhaps the capacity of Bufo
tadpoles to increase their thermal tolerance or heat
harden would be compromised during metamorphosis
compared to Xenopus tadpoles.
2. Materials and methods
2.1. Animals
Larval and postmetamorphic (juvenile) B. americanus
were collected in and around Krause Pond, Bennington,
Vermont, US, in June and July of 1991 and 1992.
X. laevis tadpoles were obtained from Nasco Biological
Supply from August 1998 to May 1999. All animals
were maintained at room temperature (20–22 C) with a
12-h photoperiod centered at noon (EST). Previously
untreated X. laevis tadpoles that transformed in the lab
were tested as juveniles. All tadpoles were maintained in
aquaria with aged tap water. B. americanus tadpoles
were fed boiled lettuce and X. laevis tadpoles were
fed commercial Xenopus tadpole nutrient. Juvenile
B. americanus were maintained in watch glasses on
moist paper towel and were fed flightless fruit flies.
Transformed X. laevis were kept in aquaria and were
fed commercial food pellets. Most animals were tested
between 8 and 14 days of capture or receipt. Three
Bufo tadpoles of the earliest stage used (see below) were
tested after only 3 days in order to ensure that they had
not developed into a later stage. However, Lutterschmidt and Hutchison (1997) note that acclimation is
rapid in ectotherms and occurs within a few days.
All experiments were performed between 1000 and
1500 h EST.
2.2. Determination of thermal tolerance
We used the CTMax as the index of thermal
tolerance, defined as the temperature at which locomotor activity becomes so disorganized that animals are
unable to escape from lethal conditions (Cowles and
Bogert, 1944). Test animals typically exhibited lower
levels of heat incapacitation such as listing and loss of
righting ability. Lutterschmidt and Hutchison (1997)
argue that onset of spasms (OS) is a preferred CTMax
endpoint. However, they were reporting on adult
ectotherms. As in fish, OS is difficult to observe reliably
in tadpoles (see Cupp, 1980; Sherman, 1980; Menke and
Claussen, 1982; Floyd, 1983; Skelly and Freidenburg,
2000). We found that the most consistent endpoint of
thermal incapacitation for all stages of tadpoles was an
abrupt cessation of movement, even when prodded, and
the temperature at which that occurred was designated
as the CTMax. Other studies of thermal tolerance in
tadpoles also use abrupt cessation of movement as the
endpoint, permitting appropriate comparisons with our
data (Cupp, 1980; Sherman, 1980; Menke and Claussen,
1982; Floyd, 1983; Skelly and Freidenburg, 2000).
CTMax determinations followed the methods of
Sherman (1980). One or two individuals (depending on
size) were placed in a beaker of 500 ml of aged tap water
at room temperature (20–22 C) and permitted to adjust
for 15–20 min prior to heating. Then the water was
heated on a hot plate at 1 C min1, a rate at which there
would be no lag between ambient temperature and deep
body temperature (Hutchison, 1961). The water was
aerated continuously and stirred with a Fisher Scientific
Digital Thermometer (calibrated to nearest 0.1 C)
which insured complete mixing of the water. As heating
progressed, tadpoles were prodded gently with the
thermometer to see if they responded. The temperature
at which all movement suddenly ceased was recorded as
the CTMax and the tadpoles were promptly removed to
room temperature water. If an animal did not revive in
the cool water, the CTMax had been exceeded and the
datum was discarded. Following the CTMax determination, tadpoles were staged according to Gosner (1960).
The snout-vent length of postmetamorphic individuals
was recorded.
2.3. Heat hardening experiments
A variety of treatments has been used in previous
studies to induce heat hardening in amphibians. The
temperature to which the animals are exposed, the
duration of exposure, and the interval between elevated
temperature exposure and heat hardening determination
all have been manipulated. In several studies, animals
were heated to within 2–5 C of CTMax (Easton et al.,
1987; Spotila et al., 1989; Yu et al., 1998). The CTMax
of tadpoles, however, changes throughout development
and a temperature of 2 C below the CTMax of
premetamorphic tadpoles might exceed the CTMax of
metamorphosing individuals (Sherman, 1980). Moreover, the CTMax of different species is different (Cupp,
1980), so we could not use one temperature for both
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E. Sherman, D. Levitis / Journal of Thermal Biology 28 (2003) 373–380
375
2.4. Statistics
species. Conversely, we wanted to use one pretreatment
temperature for all animals of the same species rather
than, say 2 C below CTMax for each stage. It would be
more ecologically significant to compare the responses
of animals of different stages to the same heat stress
(Lutterschmidt and Hutchison, 1997).
Our pilot studies indicated that a pretreatment of
10 min at 2–3 C below the CTMax of metamorphosing
tadpoles (Gosner stages 44–45) followed by a 2-h
interval at room temperature (20–22 C) would reliably
induce heat hardening in most stages. In the pretreatment of B. americanus, two animals were placed in
500 ml of aerated aged tap water at room temperature
and then heated at 1 C min1. They were held at 37
(70.5) C for 10 min and then placed back in room
temperature water. The pretreatment of X. laevis was the
same except that animals were held at 32 (70.5) C.
Following a 2-h interval at room temperature, the
CTMax of the animals was determined as above. The
stage (tadpoles) or snout-vent length (juveniles) of
pretreated animals was recorded following the experiments. Heat hardening was measured as an increase in
the mean CTMax of pretreated animals of a particular
stage or stage group compared to the mean CTMax of
non-pretreated control animals of the same stage or
stage group.
We tested B. americanus tadpoles from Gosner stages
32–46 and X. laevis tadpoles from stages 26–46. X. laevis
tadpoles metamorphosed at a much larger size than
B. americanus tadpoles. The Bufo juveniles tested were
1.0–1.4 cm long while Xenopus juveniles were 2.5–4.0 cm
long.
In order to assess the effect of heat pretreatment on
tadpoles of different stages, we applied an ANOVA
model for each species with treatments nested within
stages using the General Linear Model of SYSTAT 9
(SPSS, Inc.). In some cases, we further teased out the
interaction between treatment and stage by doing
pairwise Student’s-t tests comparing the mean CTMax
of control and pretreated animals within selected
developmental stage groupings.
3. Results
The CTMax of untreated (control) animals and heatpretreated animals of various stages are displayed in
Figs. 1 and 2. Treatment effects were highly significant
in both species (Bufo: po0:001; Xenopus: po0:001). The
development of anuran larvae is a continuous process
and thus the stage groupings are somewhat arbitrary but
based on descriptions of Gosner (1960), Sherman (1980),
and McDiarmid and Altig (1999). Tadpoles of stages up
to 35 are characterized by growth of the trunk and tail.
The hind limbs experience rapid growth and differentiation from stage 36 and continue to grow through stage
41. The front limbs erupt at stage 42 marking the
beginning of metamorphosis which is completed with
the resorption of the tail at stage 46.
B. americanus and X. laevis exhibited several similar
responses in this study. In both species, the CTMax
of control animals decreased during metamorphosis
42
Bufo americanus
CONTROL
Critical Thermal Maximum (°C)
PRE-TREATED
41
4
40
18
9
5
6
6
4
7
7
7
39
10
38
10
37
32-35
36-41
42-43
44-45
46
JUVENILE
Developmental Stages
Fig. 1. Mean CTMax of control (open bars) and pretreated (shaded bars) B. americanus of different developmental stages (71 SEM).
Sample size is indicated within each bar.
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376
40
CONTROL
Xenopus laevis
Critical Thermal Maximum (°C)
PRE-TREATED
39
38
8
7
13
8
8
9
37
12 15
8
12
7
9
36
8
9
35
26-28
29-35
36-41
42-43
44-45
46
JUVENILE
Developmental Stages
Fig. 2. Mean CTMax of control (open bars) and pretreated (shaded bars) X. laevis of different developmental stages (71 SEM).
Sample size is indicated within each bar.
(beginning with stages 42–43, culminating in stages
44–45) compared to earlier and postmetamorphic stages
(Figs. 1 and 2). The drop in CTMax from stages 36–41
to stages 44–45 was 1.4 C in Bufo (Fig. 1) and 2.1 C in
Xenopus (Fig. 2). In both species, tadpoles through stage
43 exhibited heat hardening (Figs. 1 and 2). Among the
larvae of both species, tadpoles of stages 42–43 exhibited
the greatest magnitude of heat hardening, 0.6 C in
B. americanus (p ¼ 0:0012) and 1.0 C in X. laevis
(p ¼ 0:007). At the completion of metamorphosis, stage
46, neither species displayed heat hardening (Figs. 1 and
2) though the CTMax of control animals had increased
compared to metamorphic individuals (stages 44–45).
Juveniles of both species exhibited significant heat
hardening, 0.8 C in B. americanus (Fig. 1; p ¼ 0:002)
and 0.4 C in X. laevis (Fig. 2; p ¼ 0:007).
Some important differences distinguish the responses
of these two species. At all stages, the CTMax values of
B. americanus were higher than those of X. laevis. The
mean CTMax of control premetamorphic B. americanus
tadpoles ranged from 40.3 C to–40.7 C (Fig. 1), while
those of premetamorphic X. laevis tadpoles ranged from
37.0 C to 37.6 C (Fig. 2). Similarly, juvenile American
toadlets had a mean CTMax of 39.9 C while that of
X. laevis was 37.2 C. However, the most profound
difference between these two species occurred during the
throes of metamorphosis (stages 44–45): X. laevis
tadpoles were still able to heat harden by 0.8 C
(Fig. 2; p ¼ 0:013), while the CTMax of pretreated B.
americanus actually decreased by 1.2 C (Fig. 1;
p ¼ 0:028).
Typically, animals of both species recovered from
CTMax determinations within a minute of being placed
in room temperature water. During CTMax tests using
tadpoles with distinct tails, hind limbs, and forelimbs
exposed (stages 42–44), the limbs of the tadpoles (i.e.
‘‘adult parts’’) stopped moving at lower temperatures
than the tails (i.e. ‘‘larval parts’’) in both species.
However, metamorphosing Bufo tadpoles sometimes
required up to 4 h in cool water to recover, while
metamorphosing Xenopus tadpoles recovered immediately.
4. Discussion
Comparisons of ontogenetic changes in thermal
tolerance of anurans among different studies are somewhat problematic given the different treatment regimes
in different studies. Nevertheless, the pattern of ontogenetic change in thermal tolerance exhibited by
B. americanus and X. laevis in our study is similar to
that reported in other studies. Thermal tolerance
decreases during metamorphosis compared to the
thermal tolerance of premetamorphic larvae (Table 1),
and this pattern is likely common for anurans. In studies
in which tadpole groupings distinguish among premetamorphic tadpoles (through stage 41), tadpoles just
entering metamorphosis (following the eruption of
forelimbs, stages 42–43) and tadpoles in metamorphic
climax (stages 44–45), the decrease in CTMax begins
during stage 42 and continues through stage 45. The
reported decrease in CTMax from premetamorphic
to metamorphosing tadpoles ranges from over 4 C in
B. w. fowleri and B. marinus to roughly 0.5–2 C
in B. americanus, X. laevis, B. terrestris and R. pipiens,
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377
Table 1
Change in mean CTMax ( C) of anurans from premetamorphic stages to metamorphic stages. The various acclimation temperatures
and stage comparisons are presented from different studies
Species
Bufonidae
B. terrestris
B. americanus
B. w. fowleri
B. marinus
Ranidae
R. pipiens
R. sylvatica
R. catesbeiana
Hylidae
Pseudacris triseriata
Microhylidae
Gastrophryne carolinensis
Pipidae
X. laevis
Acclimation
temperature ( C)
Premetamorphic
stages
Metamorphic
stages
Change in mean
CTMax ( C)
Study
22
27
20
20–22
20–22
30
21.8–24
20
30
25
30
35–39
35–39
42
36–41
42–43
42
42–43
42
42
40
40
40–44
40–44
44
44–45
44–45
43–44
44
43–44
43–44
44
44
0.5
0.7
1.93
1.4
0.7
0.73
4.1
0.67
1.15
3.9
5.3
Noland and Ultsch (1981)
Noland and Ultsch (1981)
Cupp (1980)
Present study
Present study
Cupp (1980)
Sherman (1980)
Cupp (1980)
Cupp (1980)
Floyd (1983)
Floyd (1983)
22
27
20
30
25
35–39
35–39
42
42
28–35
40–44
40–44
43–44
43–44
43–44
0.5
0
0.73
0.57
1.51
Noland and Ultsch (1981)
Noland and Ultsch (1981)
Cupp (1980)
Cupp (1980)
Menke and Claussen (1982)
20
30
42
42
43–44
43–44
0.19
1.09
Cupp (1980)
Cupp (1980)
20
30
42
42
43–44
43–44
1.53
2.89
Cupp (1980)
Cupp (1980)
20–22
20–22
36–41
42–43
44–45
44–45
2.1
0.9
Present study
Present study
and R. sylvatica (Table 1). While the various species
exhibited decreases of different magnitudes, these
reported differences may be related to the varying
treatment regimes and groupings of developmental
stages in the different studies.
There appear to be several interrelated predictors of
tadpole CTMax including latitude, time of year at which
larvae appear, and relative permanence of larval ponds,
all of which affect the environmental temperatures to
which tadpoles are exposed. Bufonids, which can live in
temporary ponds, typically exhibit higher CTMax than
sympatric ranids that reproduce in more permanent
ponds (Noland and Ultsch, 1981; Ultsch et al., 1999).
Predictably, bufonids are found at temperatures closer
to their CTMax than are ranids, which may facilitate
rapid metamorphosis from transient ponds (Sherman,
1980; Noland and Ultsch, 1981). Ponds in the tropics
and subtropics exhibit smaller temperature variations
than temperate ponds (Ultsch et al., 1999) and indeed
the values of CTMax of B. americanus which we
report,are roughly 3 C higher than X. laevis at all stages
of development. Bufonid tadpoles have been reported in
water of temperatures as high as 42 C (Sherman, 1980)
but data on larval ecophysiology of X. laevis are
lacking.
Cupp (1980) hypothesized that the decrease in
CTMax during metamorphosis represents an adaptive
shift downward in the thermal physiology of anurans in
anticipation of the lower temperatures to which they will
be exposed as adults. However, the increase in CTMax
following metamorphosis reported here in X. laevis and
B. americanus juveniles and in B. w. fowleri juveniles and
adults (Sherman, 1980) supports Sherman’s notion that
metamorphosis is a stressful condition due to extensive
differentiation and metamorphosing animals are simply
unable to mobilize mechanisms to cope with high
temperatures. Moreover, the thermal environment of
adult and larval X. laevis is likely to be the same as they
are found in the same ponds (Tinsley et al., 1996),
although there are no data describing the different
microenvironments that these animals might select.
The effect of heat pretreatment on thermal tolerance
of animals of different stages is summarized in Fig. 3.
Premetamorphic tadpoles of both X. laevis and
B. americanus exhibited a significant capacity for heat
hardening (Fig. 3). This capacity would be adaptive for
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378
Change in CTMax due to heat pretreatment (°C)
1.5
Bufo americanus
Xenopus laevis
1
0.5
0
-0.5
-1
-1.5
< 35
36-41
42-43
44-45
Developmental Stages
46
JUVENILE
Fig. 3. Effect of heat pretreatment on thermal tolerance of B. americanus and X. laevis of different stages. Points represent differences
between mean CTMax of heat-pretreated animals and control animals of a given developmental stage (data from Figs. 1 and 2). Points
above the 0 line reflect heat hardening except for stage 46, in which the difference between the mean CTMax of control and pretreated
animals was not significant (Bufo: p ¼ 0:19; Xenopus: p ¼ 0:20). Heat pretreatment of Bufo stages 44–45 resulted in a significant
decrease in thermal tolerance (p ¼ 0:028; see text). Earliest stages studied were 32 for Bufo and 26 for Xenopus.
tadpoles facing daily fluctuations in pond temperatures.
Typically, amphibians experiencing heat hardening
manifest an increase in thermal tolerance of 1 C or less
(Rome et al., 1992). In the only other study of heat
hardening in tadpoles of which we are aware, Menke
and Claussen (1982) reported an increase in thermal
tolerance of 0.4 C in premetamorphic (stages 28–35)
Rana catesbeiana, though their methods differed from
ours considerably. Nevertheless, in our study premetamorphic tadpoles of B. americanus of stages 32–35 and
premetamorphic tadpoles of X. laevis stages 29–35
exhibited heat hardening of 0.4 C as well (Fig. 3).
Within each species, the tadpoles that exhibited the
greatest degree of heat hardening were individuals of
stages 42–43 (Fig. 3; 0.6 C in B. americanus and 1.0 C
in X. laevis). Similarly, in their study of R. catesbeiana
tadpoles, Wollmuth and Crawshaw (1988) noted that
early metamorphic tadpoles tended to select the highest
temperatures of any stages. In order to minimize the
amount of time spent in metamorphosis, their most
vulnerable period (Wassersug and Sperry, 1977; Ultsch
et al., 1999), it might be advantageous for tadpoles to
select warmer temperatures (Wollmuth and Crawshaw,
1988) and be capable of enduring those temperatures by
heat hardening. However, Wollmuth and Crawshaw
(1988) also noted a decrease in selected temperature
during late metamorphosis and suggested that this might
be characteristic of anuran development.
In our study, late metamorphic X. laevis tadpoles
(stages 44–45) though having a lower thermal tolerance
than premetamorphic animals, still were able to heat
harden (Fig. 3). However, in what may be the most
striking result of our study, the thermal tolerance of late
metamorphic B. americanus tadpoles actually decreased
by 1.2 C following heat pretreatment (Fig. 3). The
decrease in thermal tolerance of both species during
metamorphic climax suggests that metamorphosis is
indeed stressful. The fact that metamorphosing
B. americanus tadpoles fail to heat harden and, in fact,
exhibit a lower thermal tolerance following heat
pretreatment, suggests that metamorphosis might be
more stressful for B. americanus than X. laevis. Moreover, metamorphosing American toad tadpoles required
up to 4 h to recover following CTMax determinations,
while comparable stage Xenopus tadpoles recovered
immediately.
Wassersug (1996) has argued that Xenopus has a less
extensive and less abrupt metamorphosis compared to
neobatrachians such as bufonids. Xenopus remains
aquatic as an adult. It has functional lungs through
most of its larval life (Wassersug and Feder, 1983), a
comparatively long larval period, and a protracted
metamorphosis of roughly 14 d (Wassersug and Sperry,
1977). By contrast, B. americanus undergoes more
extensive differentiation at metamorphosis (Wassersug
and Hoff, 1982). It acquires, for example, functional
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E. Sherman, D. Levitis / Journal of Thermal Biology 28 (2003) 373–380
lungs only upon metamorphosis (Wassersug and Feder,
1983). Yet the radical shift in its morphology and
physiology necessary to accommodate the change from
an aquatic to terrestrial habitat occurs during a
metamorphic climax as brief as 5 or 6 d (Sherman,
personal observation). The more extensive and abrupt
differentiation of B. americanus during metamorphosis
is reflected not simply in its inability to heat harden but,
perhaps more significantly, in its decrease in thermal
tolerance following a heat stress (Fig. 3).
Individuals of both species that had just completed
metamorphosis (stage 46) did not exhibit heat hardening
(Figs. 1 and 2). Compared to metamorphosing animals,
this represents a recovery of thermal resistance in
B. americanus as the thermal tolerance of pretreated
animals remained the same rather than decreased.
However, the significance of the inability of stage 46
X. laevis to heat harden is unclear given that animals in
metamorphic climax were able to heat harden (Fig. 2).
Among B. americanus, the developmental stage
exhibiting the greatest degree of heat hardening is the
juvenile (Fig. 3). Bufonid toadlets are heliothermic
(Sherman, 1980) and the capacity to heat harden is
likely to be beneficial given that such behavior enhances
their growth rate and shortens their time to adult size
(Lillywhite et al., 1973).
Comparisons of the thermal physiology of B. americanus and X. laevis reported here must be made
cautiously as we used individuals from a wild population
of B. americanus and laboratory reared (for many
generations) X. laevis. Moreover, a great deal is known
about the physiological ecology of bufonid larvae, while
there is little comparable literature for wild Xenopus
larvae.
379
established in several species of salamanders in which it
has been studied (Easton et al., 1987; Spotila et al., 1989;
Yu et al., 1998). Nevertheless, an investigation of levels
of constitutive hsps and hsp synthesis among tadpoles of
different stages and species might shed light on the
mechanisms underlying the ontogenetic changes in their
thermal physiology. Moreover, our observation that the
tail seems to have a higher thermal tolerance than the
legs of the same metamorphosing individual suggests
that there might be differences in constitutive hsps
between ‘‘adult parts’’ and ‘‘tadpole parts’’ within the
same individual. While hsp synthesis in adult and
embryonic amphibians has been reported, studies of
hsp synthesis in anurans undergoing metamorphosis are
entirely lacking. Finally, Denver (1997) reported that
circulating levels of corticosteroid stress hormones
increase at metamorphosis in anurans. The relationship
among corticosteroids, hsps, and thermal tolerance of
metamorphosing tadpoles is unknown and deserves
further investigation.
Acknowledgements
We would like to thank Richard Wassersug, R.C.
Tinsley, and Kerry Woods for helpful comments on
earlier drafts of this paper. Conversations with Richard
Wassersug, in particular, helped us to consider some of
the broader connections of this work. Additionally,
Kerry Woods provided invaluable statistical assistance.
This study was supported in part by funds of the
National Science Foundation, OSR-9350540 to ES.
References
4.1. Future questions
We hypothesize that the more abrupt and extensive
the differentiation of a metamorphosing anuran, the
more compromised will be its ability to heat harden
during that transformation. Of course, it is difficult to
draw phylogenetic conclusions based only on two
species and further studies of thermal tolerance during
metamorphosis of different species are warranted. Since
the 1980s, little attention has been paid to the
significance of ontogenetic change in thermal tolerance
of anurans. But studies of thermo-physiological correlates of metamorphosis among different amphibian taxa
may shed light on fitness consequences of such
transformations (see Angilletta et al., 2002).
The mechanisms underlying heat hardening in tadpoles are not known. Increased cellular thermal tolerance following a brief heat stress has been correlated
with increased synthesis of heat shock proteins
(hsps) (Feder, 1999). However, a causal relationship
between hsp synthesis and heat hardening has not been
Angilletta Jr., M.J., Niewiarowski, P.H., Navas, C.A., 2002.
The evolution of thermal physiology. J. Therm. Biol. 27,
249–268.
Cowles, R.B., Bogert, C.M., 1944. A preliminary study of
thermal requirements of desert reptiles. Bull. Am. Mus. Nat.
HisT. 83, 261–296.
Cupp Jr., P.V., 1980. Thermal tolerance of five salientian
amphibians during development and metamorphosis. Herpetologica 36, 234–244.
Denver, R.J., 1997. Proximate mechanisms of phenotypic
plasticity in amphibian metamorphosis. Am. Zool. 37,
172–184.
Dupre, R.K., Petranka, J.W., 1985. Ontogeny of temperature
selection in larval amphibians. Copeia 1985, 462–467.
Easton, D.P., Rutledge, P.S., Spotila, J.R., 1987. Heat shock
protein induction and induced thermal tolerance
are independent in adult salamanders. J. Exp. Zool. 241,
263–267.
Feder, M.E., 1999. Organismal, ecological, and evolutionary
aspects of heat-shock proteins and the stress response:
established conclusions and unresolved issues. Am. Zool.
39, 857–864.
ARTICLE IN PRESS
380
E. Sherman, D. Levitis / Journal of Thermal Biology 28 (2003) 373–380
Floyd, R.B., 1983. Ontogenetic change in the temperature
tolerance of larval Bufo marinaus (Anura: Bufonidae).
Comp. Biochem. Physiol. 75A, 267–271.
Gosner, K.L., 1960. A simplified table for staging anuran
embryos and larvae with notes on identification. Herpetologica 16, 183–190.
Hutchison, V.H., 1961. Critical thermal maxima in salamanders. Physiol. Zool. 34, 92–125.
Lillywhite, H.B., Licht, P., Chelgren, P., 1973. The role of
behavioral thermoregulation in the growth energetics of the
toad, Bufo boreas. Ecology 54, 375–383.
Lutterschmidt, W.I., Hutchison, V.H., 1997. The critical
thermal maximum: history and critique. Can. J. Zool. 75,
1561–1574.
McDiarmid, R.W., Altig, R., 1999. Research: materials and
techniques. In: McDiarmid, R.W., Altig, R. (Eds.), Tadpoles: the Biology of Anuran Larvae. University of Chicago
Press, Chicago, IL, pp. 7–23.
Menke, M.E., Claussen, D.L., 1982. Thermal acclimation and
hardening in tadpoles of the bullfrog, Rana catesbeiana.
J. Therm. Biol. 7, 215–219.
Noland, R., Ultsch, G.R., 1981. The roles of temperature and
dissolved oxygen in microhabitat selection by tadpoles of a
frog (Rana pipiens) and a toad (Bufo terrestris). Copeia
1981, 645–652.
Rome, L.C., Stevens, E.D., John-Alder, H.B., 1992. The
influence of temperature and thermal acclimation on
physiological function. In: Feder, M.E., Burggren, W.W.
(Eds.), Environmental Physiology of the Amphibians.
University of Chicago Press, Chicago, IL, pp. 183–205.
Sherman, E., 1980. Ontogenetic change in the thermal tolerance
of the toad Bufo woodhousii fowleri. Comp. Biochem.
Physiol. 65A, 227–230.
Skelly, D.K., Freidenburg, L.K., 2000. Effects of beaver on the
thermal biology of an amphibian. Ecology Lett. 3, 483–486.
Spotila, J.R., Standora, E.A., Easton, D.P., Rutledge, P.S.,
1989. Bioenergetics, behavior, and resource partitioning in
stressed habitats: biophysical and molecular approaches.
Physiol. Zool. 62, 253–285.
Tinsley, R.C., Loumont, C., Kobel, H.R., 1996. Geographical
distribution and ecology. In: Tinsley, R.C., Kobel, H.R.
(Eds.), The Biology of Xenopus. Clarenden Press, Oxford,
pp. 35–59.
Ultsch, G.R., Bradford, D.F., Freda, J., 1999. Physiology: coping with the environment. In: McDiarmid,
R.W., Altig, R. (Eds.), Tadpoles: the Biology of Anuran
Larvae. University of Chicago Press, Chicago, IL,
pp. 189–214.
Wassersug, R., 1996. The biology of Xenopus tadpoles. In:
Tinsley, R.C., Kobel, H.R. (Eds.), The Biology of Xenopus.
Clarenden Press, Oxford, pp. 195–211.
Wassersug, R.J., Sperry, D.G., 1977. The relationship of
locomotion to differential predation on Pseudacris triseriata
(Anura: Hylidae). Ecology 58, 830–839.
Wassersug, R.J., Hoff, K., 1982. Developmental changes in the
orientation of the anuran jaw suspension. A preliminary
exploration into the evolution of anuran metamorphosis.
Evol. Biol. 15, 223–246.
Wassersug, R.J., Feder, M.E., 1983. The effects of aquatic
oxygen concentration, body size and respiratory behaviour on the stamina of obligate (Bufo americanus)
and facultative air-breathing (Xenopus laevis and
Rana berlandieri) anuran larvae. J. Exp. Biol. 105,
173–190.
Wollmuth, L.P., Crawshaw, L.I., 1988. The effect of development and season on temperature selection in bullfrog
tadpoles. Physiol. Zool. 61, 461–469.
Yu, Z., Dickstein, R., Magee, W.E., Spotila, J.R., 1998. Heat
shock response in the salamanders Plethodon jordani and
Plethodon cinereus. J. Therm. Biol. 5, 259–265.